Stem cells are capable of both self-renewal and producing numerous differentiated cells. The Piwi subfamily of Argonaute proteins (herein called Piwi proteins) is required for stem cell maintenance in diverse organisms, yet its molecular function is not well understood. Piwi proteins associate with a distinct class of small RNAs, piRNAs, and modulate gene expression at both epigenetic and post-transcriptional levels. This proposal will take advantage of Hydra as a model system to further understand piwi functions in stem cells that are conserved across phylogeny. The Hydra, a basal metazoan, has a well-characterized stem cell lineage, the 1-cell lineage, in which two piwi homologs are specifically expressed. Transgenic Hydra that express GFP in the 1-cell lineage allow for in vivo observation of stem cells, thus, for the first time, the effect of piwi perturbation on stem cell behavior will be observed in the context of a living organism. In addition, because Hydra propagate clonally it is easy to obtain large, genetically pure populations for biochemical studies. Thus, the molecular function of piwi in stem cells will be investigated by characterizing PlWl-containing protein complexes and identifying targets of piwi-directed gene regulation. Finally, Hydra has a large population of putative piRNAs of which the identity and function will be explored. Importantly, Cnidarian gene sets, such as Hydra, exhibit the same complexity found in vertebrate genomes. Thus, functional discoveries made in these relatively simple metazoans will be applied to more complex organisms. These studies will advance our understanding of the conserved molecular functions of piwi in stem cells, thus contributing to the basic understanding of stem cell molecular circuitry required to manipulate stem cells for use in therapy. PUBLIC HEALTH RELEVANCE: Stem cells both self-renew and give rise to differentiated cell types of the body and therefore have great therapeutic potential in replacing lost or damaged tissues. The piwi genes are required for stem cell self-renewal in diverse organisms; they encode proteins that associate with small RNAs to modulate gene expression at many levels. Hydra, with its accessibility for manipulating stem cells in the context of a live organism, provides an effective model to explore molecular mechanisms mediated by Piwi proteins in regulating stem cell self-renewal and differentiation.